UNCONTROLLED COPY

SAM 118.05

Page 1 of 10

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 118

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

Date: October 16, 2014

Number: SAM 118.05

Supersedes: SAM 118.04, September 9, 2011

Standard Requirement: 9 CFR 113.318

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 26Nov14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 09Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 10Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 2 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 3 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro test method which utilizes viral 

plaque forming units (PFU) in a cell culture system to titer pseudorabies virus (PRV) in 

modified-live vaccines.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 36°± 2°C, 5% ± 1% CO2, high humidity incubator (Model 3158, Forma 

Scientific Inc.)

2.1.2 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries Inc.)

2.1.3 Blender

2.1.4 Micropipettors: 200-μL and 1000-μL single channel

2.1.5 Water bath

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 PRV Reference Virus, Shope strain

2.2.2 Madin-Darby bovine kidney (MDBK) or other permissive cells

2.2.3 Minimum Essential Medium (MEM)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3)

3. Q.S. to 1000 mL with deionized water (DI), adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl).

4. Sterilize through 0.22-μm filter.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 4 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

5. Aseptically add:

a. 10 mL L-glutamine (200 mM)

b. 5 mL lactalbumin hydrolysate or edamine

c. 50 μg/mL gentamicin sulfate

6. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL of MEM

2. Aseptically add 100 mL gamma-irradiated fetal bovine serum (FBS).

3. Store at 2°- 7°C.

2.2.5 2X Medium

1. 100 mL 10X MEM with Earles salts without bicarbonate

2. 2.2 g sodium bicarbonate

3. 340 mL DI

4. Sterilize through 0.22-μm filter.

5. Aseptically add:

a. 50 μg/mL gentamicin sulfate

b. 50 mL FBS

6. Store at 2°- 7°C.

2.2.6 2% Tragacanth Gum (Trag)

1. 20 g Trag

2. 1000 mL DI

3. Mix vigorously small amounts at a time with a blender set on high.

4. Pour 500 mL each into 1000-mL media bottles.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 5 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

5. Sterilize by autoclaving at 15 psi for 30 minutes.

6. Store at 2°- 7°C.

2.2.7 Overlay Medium

1. Mix equal volumes of 2X Medium and 2% Trag.

2. Store at 2°- 7°C.

2.2.8 70% Ethyl Alcohol

1. 74 mL ethyl alcohol

2. 26 mL DI

3. Store at room temperature. 

2.2.9 Crystal Violet Stain

1. 7.5 g crystal violet

2. 50 mL 70% ethyl alcohol

3. Dissolve crystal violet in alcohol.

4. 250 mL formaldehyde

5. Q.S. to 1000 mL with DI, filter through filter paper.

6. Store at room temperature.

2.2.10 Tissue culture plates, 6-well

2.2.11 12 x 75-mm polystyrene tubes

2.2.12 Graduated cylinders, 25-, 50-, 100-, and 250-mL, sterile

2.2.13 Serological pipette, 10-mL
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 6 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in the immunologic basis of virus titration assays, cell 

culture maintenance, and in the principles of aseptic techniques.

3.2 Preparation of equipment/instrumentation

Set the water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 Two days prior to test performance

Seed 6-well tissue culture plates with MDBK cells, in Growth Medium, at a cell 

count that will produce a monolayer after 2 days of incubation at 36°± 2°C. 

These become the MDBK plates. Growth Medium is changed if excess acidity of 

the medium is observed as indicated by a change from red to yellow of Growth 

Medium.

3.3.2 On day of test performance

Rapidly thaw a vial of PRV Reference Virus in a water bath at 36°± 2°C. Dilute 

the virus in 4.5 mL MEM to contain 15-40 PFU/100 μL.

3.4 Preparation of the sample (on day of test performance)

3.4.1 Rehydrate a vial of the Test Vaccine by removing the seal and stopper 

from both the Test Vaccine bottle and the bottle containing the accompanying 

diluent. Measure the diluent into a sterile graduated cylinder according to the 

number of doses indicated on the manufacturer’s instructions (e.g., for a 50-dose 

container of 2-mL-per-dose, reconstitute with 100 ml of diluent), and aseptically 

pour the diluent into the lyophilized bottle of vaccine. Mix by vortexing. Allow to 

sit for 15 ± 5 minutes at room temperature.

3.4.2 Prepare serial tenfold dilutions of test vaccine. Serial tenfold dilutions may 

be made as follows:

1. Place 4.5 mL of MEM with the pipette into labeled 12 x 75-mm 

polystyrene tubes.

2. Pipette 500 μL of test vaccine to the 10-1tube, mix by vortexing. 

Discard pipette tip.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 7 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

3. Repeat Step 2 to the remaining tubes. Continue as needed (10-2to 10-

5

), transferring 500 μL from previous tube to the next dilution.

4. Performance of the Test

4.1 Decant the Growth Media from MDBK plates.

4.2 Pipette 100 μL/well from each dilution of test vaccine to 2 wells of a MDBK 

plate. Mix by gentle swirling.

4.3 Pipette 100 μL/well of the diluted Reference Virus Control to 2 wells of 1 MDBK 

plate. Mix by gentle swirling.

4.4 Maintain 2 or more wells as uninoculated cell culture controls.

4.5 Incubate inoculated plates at 36°± 2°C in a CO2 atmosphere for 60 ± 10 minutes

for virus adsorption.

4.6 Add 3.0 mL/well of warmed (36o) Overlay Medium (see Section 2.2.7) to the 

plates. Discard any unused, warmed Overlay Medium. 

4.7 Incubate the MDBK plates undisturbed at 36°± 2°C in a CO2 atmosphere for 4 

days.

4.8 At the end of incubation, decant Overlay Medium. Pipette 2 mL of the Crystal 

Violet Stain (see Section 2.2.9) into each well of the plates using the repetitive syringe.

4.9 Allow plates to stand at room temperature for 15 ± 5 minutes.

4.10 Discard the crystal violet stain down a sink. Wash the cell monolayers by dipping 

each plate several times in a container of running water from the cold faucet. Allow to air 

dry.

4.11 PFU counting

4.11.1 The PFU are visible as clear, circular areas in the cell monolayer where 

the cells have been destroyed by the virus.

4.11.2 Count the number of PFU for each well.

1. Average the number of PFU between the duplicate wells for each test 

vaccine dilution.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 8 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

2. Average the number of PFU between the 2 wells of the Reference 

Virus Control wells.

3. Determine the virus titers and express as PFU/dose. Take the dilution 

of a test vaccine that contains an average of at least 30 PFUs. 

Example:

Log10 of plaque count (30) 1.48

Log10 of dilution counted (10-3) 3.00

Log10 of 2-mL dose factor (20) 1.30

 

Virus titer/dose (total) 5.78

The test vaccine contains 105.78 PFU/2-mL-dose.

5. Interpretation of the Test Results

5.1 The test is invalid if visible contamination is observed in all dilutions of a Test 

Vaccine.

5.2 For a valid assay, the Reference Virus Control must have an average PFU count 

between 15-40.

5.3 Any test not meeting the criteria of Sections 5.1 and 5.2 is considered a NO 

TEST and may be repeated without prejudice.

5.4 One plaque represents a single infective unit whereas the 50% endpoint infective 

dose (one ID50) is statistically equivalent to a theoretical 0.69 of an infective unit. Fifty 

percent endpoints will be 1.44 times those expressed as PFU/unit of inoculation. 

Therefore, to express PFU titer as 50% tissue culture infective dose (TCID50) titer, 

multiply the PFU by 1.44 or add 0.16 to the log10 value of the PFU titer. In the example 

above, the TCID50 would be 5.78 + 0.16 = 5.94 or 105.94 TCID50/2-mL-dose.

5.5 If the validity requirements are met and the titer of the vaccine is greater than or 

equal to the titer contained in the filed outline of production for the product under test, 

the product is considered SATISFACTORY.

5.6 If the validity requirements are met but the titer of the Test Vaccine is lower than 

the required minimum, it must be retested according to 9 CFR 113.8.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 9 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

6. Report of Test Results

6.1 Test results are reported as the virus titer in TCID50/dose.

6.2 Record all test results on the test record. 

7. References

7.1 Title 9, Code of Federal Regulations, U.S. Government Printing Office, 

Washington, DC.

7.2 Conrath TB.: Handbook of Microtiter Procedures. 1972. Clinical and Research 

Applications Laboratory, Cooke Engineering Company, Alexandria, VA.

8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .04

 The phrase "available from the Center for Veterinary Biologics/CVB" has been 

removed from the document as these reagents are no longer supplied by the CVB.

Version .03

 The Contact information has been updated.

 The document number has been changed from VIRSAM0118 to SAM 118.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 2.1.7 “Self-refilling syringe” has been removed.

 2.2.3.2 The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g 

to 1.1 g.
Center for Veterinary Biologics SAM 118.05

Testing Protocol Page 10 of 10

Supplemental Assay Method for Titration of Pseudorabies Virus in Vaccines

UNCONTROLLED COPY

 2.2.3.5/2.2.5.5 Penicillin and streptomycin have been deleted.

 2.2.12 “10 ml syringe and needles” has been replaced with graduated cylinders.

 3.4.1 “10 ml syringe and needle” has been deleted. Instructions have been added for

reconstituting the test vaccine with accompanying diluent by graduated cylinders

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system.